The present invention relates to molecular therapies for cancer and HIV infections in which tumor cells or immune cells are stably transduced with a variety of genes introduced by high efficiency delivery systems, such as retroviral and adenoviral vectors.
One strategy for the treatment of localized disease is to render targeted cells sensitive to normally nontoxic chemotherapeutic agents using so-called "suicide genes" such as the herpes simplex virus thymidine kinase (HSV-tk) gene, the related varicella zoster TK gene or the bacterial xanthine/guanine phosphoribosyltransferase gpt gene. HSV-tk, for example, converts the nontoxic guanosine analogue ganciclovir into a phosphorylated compound that acts as a chain terminator in DNA synthesis, selectively killing dividing cells.
The codA gene of Escherichia coli encoding cytosine deaminase (hereinafter referred to as CDase) represents another potential suicide gene to be used for the selective elimination of unwanted human cells. Cytosine deaminase is the first enzyme of the only metabolic pathway by which exogeneous cytosine or endogeneous cytosine from pyrimidine nucleotide breakdown is utilized by way of hydrolytic deamination to uracil and ammonia. Cytosine deaminases have been found in prokaryotes and lower eukaryotes but appear to be absent in higher eukaryotes, both in mammals as well as in plants Koechlin et al., Biochem Parmacol. 15, 435-446 (1966)! Ross, C. Plant Physiol. 40, 65-73 (1965)!. Cytosine deaminase also deaminates the innocuous fluorocytosine (hereinafter referred to as FC) into fluorouracil (hereinafter referred to as FU), a highly toxic compound when efficiently converted to 5-fluoro-UMP. Cells lacking cytosine deaminase activity either as a consequence of a mutational inactivation as illustrated by codA and fcyl (genes coding for cytosine deaminase) mutants of Escherichia. coli and Saccharomyces cerevisiae respectively, or because they are naturally deficient for this enzyme, as are mammalian and plant cells, are resistant to 5-fluorocytosine Kilstrup et al., J. Bacteriol 171, 2124-7 (1989)! Jund R. & Lacroute F. Journal of Bacteriology 102, 607-615 (1970)!. This property provides the basis for the use of the E.coli coda gene as a suicide or a negative selection gene in a number of recently reported experiments with mammalian and plant cells Huber et al., Cancer Res 53, 4619-26 (1993); WO 93/01281; Mullen et al., Cancer Res 54, 1503-6 (1994)! where transformed cells were shown to have acquired cytosine deaminase activity and to be sensitive to treatment with 5-fluorocytosine.
In the patent application WO 93/01281, Mullen and Blaese described a negative selection system comprising a modified bacterial gene for cytosine deaminase that has the ability to produce a toxic antimetabolite from FC in eukaryotic cells. In addition, Mullen and Blaese claimed a double negative selection based on a modified cytosine deaminase gene and the herpes thymidine kinase gene in a selection involving 5-fluorocytosine and ganciclovir an acyclic nucleoside analog of guanosine where triphosphate derivatives of ganciclovir compete with dGTP. However, it should be noted that a large majority of tumour cells are not sensitive to such a method of therapy.
The differential sensitivity to FC between parent cells and cells transfected by a cytosine deaminase gene varies greatly according to cell lines. Almost no sensitivity to fluorocytosine was observed in murine melanoma B16 clones transfected by a codA bearing vector and retained for high expression of cytosine deaminase activity (as illustrated in example 8 hereafter). The poor fluorocytosine sensitivity of cultured cells expressing high CDase activity, a situation encountered with many tumors, as reported by J. D. Harris in an oral communication at the 1994 Cold Spring Harbor Laboratory meeting on gene therapy, could be accounted for by at least two potential mechanisms.
One is related to the transport of cytosine. All natural nucleobases and their fluorinated analogs are hydrophilic and diffuse through the plasma membrane only very slowly. Their efficient permeation is dependent on special transport proteins. Fluorocytosine is taken up in E.coli by a cytosine specific permease encoded by codB. The crucial role of an active cytosine transport system in determining fluorocytosine sensitivity in this bacteria was demonstrated with mutants deleted for the codb gene. The normal situation appears to be the opposite in mammals. Although mammalian cells possess a uracil transport system, cytosine is not actively transported in various cell lines or in human erythrocytes, where entry seems to be entirely by passive diffusion Plagemann et al., Biochim Biophys Acta 947, 405-43 (1988)!.
The other mechanism is based on the existence and relative importance of anabolic and catabolic pathways that modulate fluorouracil concentration. In microorganisms, endogeneous uracil is directly converted to UMP by uracil phosphoribosyltransferase (UPRTase) and may also be converted to UMP and dUMP through the concerted action of uridine phosphorylase and uridine kinase Munch-Petersen A. & Mygind B. In. Metabolism of nucleotides, nucleosides and nucleobases in microorganisms (1983)!.
However, this two-step route operates only if either of the substrates for uridine phosphorylase, i.e. ribose-1-phosphate or desoxyribose 1-phosphate, and uracil are present at high intracellular concentrations. In animal cells, uracil and fluorouracil are metabolized in two steps to UMP and F-UMP via the uridine intermediate Mulkins M. A. & Heidelberger C. Cancer Res 42, 965-73 (1982); Schwartz et al., Biochem Pharmacol 34, 3585-9 (1985)! in absence of uridine phosphoribosyl transferase activity Kornberg A. & Baker T. In DNA replication Freeman, W. M. and Company, New York, 53-100 (1992)!. In addition, a proportion of uracil and fluorouracil are catabolized to the atoxic dihydrouracil and dihydrofluorouracil respectively.
Therefore, the toxicity of FC on cells expressing an introduced cytosine deaminase gene depends on the amount of the formed FU which is converted to 5-fluoro-UMP and, through the de novo pyrimidine pathway, to 5-fluoro-dUMP, an irreversible inhibitor of thymidylate synthase and hence of DNA synthesis by deprivation of dTTP Kornberg A. & Baker T. In DNA replication Freeman, W. M. and Company, New York, 53-100 (1992)!.
Another drug whose operation concerns the pyrimidine metabolic pathway, is Azidothymidine, for the treatment of human immuno deficiency virus (HIV)infection.
AZT is one of the primary chemotherapeutic agents used in the treatement of HIV infection. AZT readily enters cells and is converted to the nucleoside phosphates AZT-MP, AZT-DP and AZT-TP by the sequential action of thymidine kinase, thymidylate kinase and nucleoside diphosphokinase. AZT is rapidly converted to AZT-MP by thymidine kinase but the resulting AZT-MP accumulates since it is a very poor substrate for human thymidylate kinase. The concentration of AZT-MP is typically &gt;50 fold higher than that of AZT-TP in human cell lines Furman et al. Proc Natl Acad Sci USA 83, 8333-7 (1986)!. Although AZT-TP is an inefficient substrate for cellular DNA polymerases, AZT-MP is incorporated into DNA Copeland, J Biol Chem 267,21459-64 (1992)!.